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Tumor-selective drug delivery could enhance anticancer efficacy and avoid drug side effects. However, because of tumor heterogeneity, current nanoparticle-based drug delivery systems rarely improve clinical outcomes significantly, commonly only reducing systemic toxicity. In this work, a new tumor-specific, tyrosinase-responsive cascade amplification release nanoparticle (TR-CARN) was developed to fulfill the needs for tumor-specific drug delivery and high efficacy cancer treatment. Tyrosinase (Tyr) is specifically expressed in melanomas and can catalyze acetaminophen (APAP) to increase reactive oxygen species (ROS). It was therefore utilized here to initiate the ROS amplification procedure. In TR-CARN, a ROS-responsive prodrug BDOX was loaded into an amphiphilic polymer, and APAP was linked to the polymer through a ROS-cleavable thioether bond. TR-CARN caused reduced side effects during the delivery because of the low toxicity of BDOX. Once TR-CARN entered into the tumor, endogenous ROS triggered initial APAP and BDOX release. Tyr-mediated ROS synthesis by APAP then accelerated APAP and BDOX release and toxification. Consequently, TR-CARN achieved melanoma-specific treatment of high efficacy through the cascade amplification strategy with enhanced biosafety.

The affinity isolation of pre-purified plasmid DNA (pDNA) from model buffer solutions using native and poly(ethylene glycol) (PEG) derivatized zinc finger-GST (Glutathione-S-Transferase) fusion protein was examined in PEG-dextran (DEX) aqueous two-phase systems (ATPSs). In the absence of pDNA, partitioning of unbound PEGylated fusion protein into the PEG-rich phase was confirmed with 97.5% of the PEGylated fusion protein being detected in the PEG phase of a PEG 600-DEX 40 ATPS. This represents a 1322-fold increase in the protein partition coefficient in comparison to the non-PEGylated protein (Kc = 0.013). In the presence of pDNA containing a specific oligonucleotide recognition sequence, the zinc finger moiety of the PEGylated fusion protein bound to the plasmid and steered the complex to the PEG-rich phase. An increase in the proportion of pDNA that partitioned to the PEG-rich phase was observed as the concentration of PEGylated fusion protein was increased. Partitioning of the bound complex occurred to such an extent that no DNA was detected by the picogreen assay in the dextran phase. It was also possible to partition pDNA using a non-PEGylated (native) zinc finger-GST fusion protein in a PEG 1000-DEX 500 ATPS. In this case the native ligand accumulated mainly in the PEG phase. These results indicate good prospects for the design of new plasmid DNA purification methods using fusion proteins as affinity ligands.

Amphipathic pH-responsive polymers have shown to increase the permeability of cell membranes to trehalose hence improving the cryopreservation of mammalian cells. However, the trafficking of both the polymer and trehalose across the cell membrane has not yet been thoroughly analysed. The objective of this study was to investigate the effect on cryopreservation of the trafficking of the disaccharide trehalose along PP-50, an amphipathic polymer, through an osteosarcoma cell line (SAOS-2). Confocal microscopy analysis confirmed the presence of intracellular labelled trehalose only when incubated in the presence of PP-50. Further analysis confirmed that both trehalose and PP-50 localised in the cytoplasm, accumulated mainly in the perinuclear area. Quantitative analysis of the colocalisation between trehalose and PP-50 showed Pearson and Manders coefficients of 0.862 ± 0.008 and 0.766 ± 0.033, respectively, suggesting a high degree of intracellular colocalisation between these molecules. Cryopreserved cells pre-incubated with trehalose and PP-50 showed increased cryosurvival when compared with cells pre-incubated in the absence of the polymer. PP-50 showed to be directly involved in the uptake of trehalose, a critical characteristic towards use in cryopreservation and biomedical applications.

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required. © 2007 Wiley Periodicals, Inc.

We report the encapsulation of the hydrophilic model molecule calcein in the Zr-based MOF UiO-66, followed by amorphization of the framework by ball-milling. We show controlled release of calcein over more than 30 days, compared with the 2 day release period from crystalline UiO-66.

Carbon nanotube salts prepared by treating single-wall carbon nanotubes (SWNTs) with lithium in liquid ammonia react with ω-bromocarboxylic acids to yield sidewall-derivatized SWNTs. These can be reacted with amine-terminated polyethyleneglycol (PEG) chains (e.g., H2N−mPEG−OMe, MW = 5000 Da) to yield water-soluble PEGylated SWNTs. Short nanotubes (average length ∼ 60 nm) functionalized by PEGylation of the carboxylic acid groups at the end of the nanotubes exhibit greater solubility in water than the longer (average length ∼ 300 nm) sidewall-PEGylated pristine nanotubes.

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction. © 2006 Wiley Periodicals, Inc.

The development of a protein-mediated dual functional affinity adsorption of plasmid DNA is described in this work. The affinity ligand for the plasmid DNA comprises a fusion protein with glutathione-S-transferase (GST) as the fusion partner with a zinc finger protein. The protein ligand is first bound to the adsorbent by affinity interaction between the GST moeity and gluthathione that is covalently immobilized to the base matrix. The plasmid binding is then enabled via the zinc finger protein and a specific nucleotide sequence inserted into the DNA. At lower loadings, the binding of the DNA onto the Fractogel, Sepharose, and Streamline matrices was 0.0078 +/- 0.0013, 0.0095 +/- 0.0016, and 0.0080 +/- 0.0006 mg, respectively, to 50 microL of adsorbent. At a higher DNA challenge, the corresponding amounts were 0.0179 +/- 0.0043, 0.0219 +/- 0.0035, and 0.0190 +/- 0.0041 mg, respectively. The relatively constant amounts bound to the three adsorbents indicated that the large DNA molecule was unable to utilize the available zinc finger sites that were located in the internal pores and binding was largely a surface adsorption phenomenon. Utilization of the zinc finger binding sites was shown to be highest for the Fractogel adsorbent. The adsorbed material was eluted with reduced glutathione, and the eluted efficiency for the DNA was between 23% and 27%. The protein elution profile appeared to match the adsorption profiles with significantly higher recoveries of bound GST-zinc finger protein.

The preparation, characterisation and testing of stable non-porous coated perfluorocarbon supports functionalised with the metal chelate, iminodiacetic acid (IDA) is described. Polyvinyl alcohol (PVA), a neutral hydrophilic polymer was esterified with perfluorooctanoyl chloride and anchored to the surface of solid perfluorocarbon particles through multiple fluorophilic interactions. The PVA-coated particles were then activated by epoxidation and coupled with IDA. The presence of surface-attached chelates was clearly demonstrated by the binding and selective desorption of Zn2+ ions. Three particulate perfluorocarbons were selected as potential starting materials and the conditions for preparation of metal chelating adsorbents optimised with respect to ease of manufacture, ligand density and binding capacity towards a monoclonal antibody known to bind to commercial Zn(2+)-IDA supports. The choice of base particle strongly influenced the ligand densities and specific binding capacities towards the monoclonal antibody that could be achieved under optimal preparative conditions. Possible ways in which these metal chelating adsorbents may be employed to recover the monoclonal antibody directly from culture vessels are discussed.

Magnetic cell labeling has numerous applications in cell separation and sorting, tissue engineering and magnetic resonance imaging. Mammalian cells can be magnetically labeled by first biotinylating their cell membrane proteins and then binding streptavidin‐coated paramagnetic particles onto the biotinylated proteins. Characterization studies indicated that the extent of cell biotinylation could be controlled by varying the biotinylating reagent concentration. Since the paramagnetic particles have high mass magnetization, the labeled cells were very responsive to magnetic field gradients and they could be targeted and patterned precisely using magnets. Magnetic multicellular spheroids were generated from these labeled cells and the constituent cells were found to be viable. These spheroids could be patterned using magnetic fields in a few seconds. This cell labeling methodology can be adapted to a wide variety of mammalian cell types. The labeled cells can then be manipulated in two dimensional cell cultu...

The DNA binding fusion protein, LacI-His6-GFP, together with the conjugate PEG-IDA-Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600-DEX 40 ATPS containing 0.273 nmol of LacI fusion protein and 0.14% (w/w) of the functionalised PEG-IDA-Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the LacI binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG-dextran system as a second extraction system, with 80-90% of pDNA partitioning to the bottom phase. This represents about 7.4 microg of pDNA extracted per 1 mL of pUC19 desalted lysate.

The reaction rates of ground-state OH radicals, X2Π(v″= 0), with the molecules CH4, CO, HCl, DCl, HBr and DBr have been investigated in the gas phase by time-resolved resonance fluorescence at λ= 307 nm [OH(A2∑+–X2Π, (0, 0)]. The OH radicals were generated photochemically by the repetitive vacuum ultraviolet photolysis of water vapour in a flow system, kinetically equivalent to a static system, using a high-intensity, magnetically pinched pulsed light source, and optically excited to the A2∑+ state by means of a resonance source operating in the standard orthogonal arrangement. The resulting time-resolved resonance fluorescence signals were monitored by means of a highly sensitive detection system employing pre-trigger photomultiplier gating, photon counting and signal averaging. Absolute second-order rate constants (k/cm3 molecule–1 s–1, 300 K) were obtained for the reactions of OH with the above molecules as follows: CH4(7.66 ± 0.64)× 10–15 CO (1.46 ± 0.12)× 10–13, HCl(6.66 ± 0.52)× 10–13, DCl (3.48 ± 0.30)× 10–13, HBr (6.01 ± 0.32)× 10–12 and DBr (2.05 ± 0.14)× 10–12, the quoted errors being 2σ. The rate constants for CH4 and CO constituted a kinetic test of the system, being in accord with accepted values for these quantities. The data for the hydrogen halides are compared, where possible, with previously determined rates obtained from monitoring OH directly by various methods. To the best of our knowledge, the absolute rate constant for the reaction with DBr has not been reported hitherto from direct monitoring of OH.

Ground state antimony atoms, Sb[5p3(4S)], have been studied by time-resolved resonance fluorescence at λ= 231.1 nm (64P½→ 54S) in the “single-shot” mode. The antimony atoms were generated by pulsed irradiation of SbMe3 using a “magnetically-pinched”, Garton light source with a high spectral output in the vacuum ultraviolet. Optical excitation of the resulting Sb(54S) was effected by means of a sealed microwave-powered atomic emission source, and the resonance fluorescence was monitored photoelectrically with a photomultiplier tube gated with a “pre-trigger” system. Kinetic studies of Sb(54S) in the presence of NO and of O2 are described and the resulting rate constants are compared with analogous data derived previously using time-resolved resonance absorption at the same wavelength, and discussed in terms of departures from the standard Beer–Lambert law for light absorption. Radiation trapping calculations are also described which involve summation of the Voigt profiles over all the individual nuclear hyperfine components that contribute to the λ= 231.1 nm transition. Solutions of the diffusion equation for radiation with different boundary conditions are employed in the calculations of the relationship between fluorescence intensity and particle density. The results are compared with the experimental fluorescence intensity calibration for this system.

We present a kinetic study of ground state arsenic atoms, generated photochemically and monitored by fluorescence in the time-resolved mode following optical excitation. As[4p3(4S)] was derived from the repetitive pulsed irradiation of low pressures of either AsMe3 or AsCl3, in the presence of excess helium, in a flow system which is kinetically equivalent to a static system. The transient atoms were then monitored photoelectrically by summation of the optically excited fluorescence, principally at the wavelengths λ= 189.04, 193.76 and 197.76 and 197.23 nm [4p25s(4P5/2, , ½)→ 4p3(4S), respectively] using a “pre-trigger” photomultiplier gating system coupled with singal averaging. The decay of the arsenic atoms in the presence of the added reactant gases O2, Cl2, NO and C2H4 is described, for which second-order reaction rate constants are reported. These data are compared with previous results derived from time-resolved resonance absorption studies at λ= 189.04 nm. The two sets of rate constants resulting from the resonance absorption fluorescence and absorption studies are then employed in order to discuss the modified Beer–Lambert Law, Itr=I0 exp [–Iµ(cl)γ], that has been used in the resonance absorption measurements. Finally, we present a model radiation trapping calculation including the effects of nuclear hyperfine interaction using the diffusion theory of radiation for the three wavelengths monitored in fluorescence, and calculate the relationship between fluorescence intensity and particle density. This is compared with empirical measurements of the fluorescence intensity against particle density calibration.

The temporal behaviour of ground state phosphorus atoms, P[3p3(4S)], generated by repetitive pulsed irradiation in a flow system, has been studied by time-resolved resonance fluorescence. P(34S) was generated photochemically from PCI3 in the presence of excess helium buffer gas and optically excited by means of the emission from a microwave-powered atomic spectroscopic source. The total resonance fluorescence in the vacuum ultraviolet from the systems P[3p24s(4P½,,5/2)→ 3p3(4S)](λ= 178.77, 178.29 and 177.50 nm, respectively) and P[3p4(4P5/2,,½)→ 3p3(4S)](λ= 167.97, 167.46 and 167.17 nm, respectively) was monitored photoelectrically and the signal-averaged results employed to characterise the kinetic decay of the phosphorus atom in the presence of various added gases. An empirical calibration of the relationship between the fluorescence intensity and the relative particle density of P(34S) was established and the linear region employed for kinetic study. Second-order rate constants (kR, cm3 molecule–1 s–1, 300 K) are reported for the reaction of P(34S) with the following gases: O2(2.1 ± 0.3 × 10–12), Cl2(3.4 ± 0.3 × 10–12), NO (5.3 ± 0.5 × 10–13), C2H2(1.2 ± 0.1 × 10–13), C2H4(6.8 ± 0.8 × 10–13) and PCl3(1.7 ± 0.3 × 10–12). These data are compared with previous results obtained by time-resolved resonance line absorption and used to consider further the modified Beer–Lambert law, Itr=I0 exp [–Iµ(cl)γ] used in the resonance absorption measurements. Finally, radiation trapping calculations are described for the systems P[3p24s(4PJ)] and P[3p4(4PJ)]→ P[(34S)] using the diffusion theory of radiation. Line shapes employing the summation of Voigt profiles for all the nuclear hyperfine components contributing to a given electronic transition are used. The calculated relationships between the fluorescence intensity and P[(34S)] for various boundary conditions of the diffusion equation are compared with the empirical calibration.

Ground state nitrogen atoms, N(24S), generated by pulsed photochemical initiation, have been, studied by time-resolved resonance fluorescence following optical excitation in the vacuum ultraviolet, λ= 120 nm, N[2p,23s(4P½,,[graphic omitted])→ 2p3(4S)]. Atomic nitrogen, principally in the optically metastable doublet states, N[2p3(2DJ, 2PJ)], was produced by the repetitive vacuum ultraviolet photolysis of N2O and collisionally quenched to the 4S state by the use of N2 as a buffer gas. Photoelectric detection of the vacuum u.v. signals employed photon counting coupled with signal summation in a multiscalar. Considerable care was required to eliminate spurious photon signals at λ= 130 nm due to O(23PJ)[O(33S1)→ O(23PJ)]. Absolute rate data for the collisional removal of N(24S) in the presence of the gases NO, NO2, O3 and C2H4 are presented and compared with previously reported data. The results for N + C2H4 are further discussed in relation to the “HCN limiting yield” method that has been employed hitherto as a marker for N atom kinetics.

We describe a kinetic investigation of H and D atoms in their electronic ground states by the method of time-resolved Lyman-α resonance fluorescence in the vacuum ultraviolet following pulsed photochemical generation of the atoms. The method involved repetitive pulsing, photon counting and signal averaging. Absolute rate constants (k/cm3 molecule–1 s–1, 300 K) are reported for the following reactants; H + H2S, (8.6 ± 0.5)× 10–13; D + D2S, (7.9 ± 0.4)× 10–13; H + HBr, (6.0 ± 0.1)× 10–12; D + HBr, (4.1 ± 0.1)× 10–12; H + DBr, (4.7 ± 0.1)× 10–12; D + DBr, (2.7 ± 0.1)× 10–12. The rates for the reactions with HBr and DBr are compared with values previously derived from e.p.r. measurements on H and D in a flow discharge system.